Modified from the Oaks Lab protocol (https://github.com/phyletica/lab-protocols/blob/master/3rad-protocol.md) and the BadDNA @ Univ. Georgia protocol (https://baddna.uga.edu/protocols.html)
Before beginning the 3RAD protocol, you will first quantify the DNA concentration of each sample (we perform this step using a Qubit fluoremeter).
Following quantification, you will then standardize your samples to 20 ng/µL.
Note: Step 2 (Ligation) must occur immediately following Step 1.
Materials Needed:
- 20,000 U/mL New England BioLabs (NEB) Restriction Enzymes
- XbaI
- EcoRI-HF
- NheI-HF
- molecular grade H2O
- 10X NEB CutSmart Buffer
- 2.5 µM Read 1 (i5) and Read2 (i7) Adapters
- a working stock will need to be made before beginning. Our lab creates several i5 adapter sets in 8-strip PCR tubes and i7 adapter sets in a 12-strip for easy grab use. DO NOT THAW UNUSED STOCK.
- Standardized DNA (20 ng/µL)
Materials Note: only take out enzymes/polymerase from freezer when they are ready to be used. Buffers for Digestion and Ligation can be thawed prior to Digestion step (at start of day)
Master Mix
| Enzyme Digestion | 1X | 112X |
|---|---|---|
| NEB CutSmart | 1.5 µL | 168 µL |
| dH2O | 3.0 µL | 336 µL |
| XbaI | 0.5 µL | 56 µL |
| EcoRI-HF | 0.5 µL | 56 µL |
| NheI-HF | 0.5 µL | 56 µL |
| Total Volume | 6 µL | 672 µL |
vortex master mix upon completion
[Semi-Skirted PCR Plate]
-
Take a strip of eight 0.5 mL PCR tubes and pipette 82 µL into each tube to use as aliquot for use of multi-channel pipetting.
-
Place 6 µL of master mix in each well using a multi-channel pipette.
-
Place 2 µL of each adapter in correct well using a multi-channel pipette (see image below for plate orientation).
- use 12-channel pipette for i7 adapters.
- use 8-channel pipette for i5 adapters.
-
Place 5 µL of DNA in each well after adding adapters using a multi-channel pipette.
-
Seal 96-well plate with adhesive film and proceed to 1b.
Per Well Total: 15 µL
- Incubate samples at 37°C for 1 hour with no heated lid.
- Spin down plate after incubation.
- Place on ice block before proceeding with Step 2.
Materials Needed:
- molecular grade H20
- Promega or NEB 10mM rATP
- 10X Ligase Buffer (takes a while to thaw, so best to start at beginning on protocol.
- 400,000 U/mL NEB T4 DNA Ligase
Master Mix
| Ligation Mix | 1X | 112X |
|---|---|---|
| dH2O | 2.75 µL | 308 µL |
| rATP | 1.5 µL | 168 µL |
| Ligase Buffer | 0.5 µL | 56 µL |
| DNA Ligase | 0.25 µL | 28 µL |
| Total Volume | 5 µL | 560 µL |
Heat Ligase Buffer to 56-60 °C to remove precipates
vortex master mix upon completion
- Take a strip of eight 0.5 mL PCR tubes and pipette 68 µL into each tube to use as aliquot for use of multi-channel pipetting.
- Using a multi-channel pipette, add 5 µL of the master mix to each well.
- Seal 96-well plate with adhesive film and proceed to 2b.
Per Well Total: 20 µL
- Incubate samples without heated lid
- 2 cycles of:
- 22 °C for 20:00
- 37 °C for 10:00
- 80 °C for 20:00
- reduce to 10 °C
- Spin down plate after incubation.
Note: While the samples incubate, make fresh 70% EtoH for Bead Cleanup & begin Kapa Hifi Buffer Thaw
Materials Needed:
- molecular grade H20
- Omega BioTek Beads
- 70% EtOH
- Low EDTA TE Buffer
- Spin down PCR product after removing from thermal cycler.
- Pool 5 µL of each ligation product into 1.5 mL tube. You can use an eight channel pipette into 8 strip tubes and then combine the 8 tubes into a 1.5 mL tube for speed.
- Mix pooled ligation product with 1X beads i.e. 480 µL of Beads and vortex + spin down.
- Sit for 5 minutes.
- Place on magnet and wait until clear.
- Carefully remove and discard the supernatent without disturbing the beads. DO NOT TOSS BEADS
- Wash with 70% EtOH.
- Add 500 µL 70% EtOH and let sit for 1 minutes on magnet.
- Pipette EtOH from tube.
- Repeat Step 4.
- Spin down and pipette up last drops of ethanol with a 0.1-10uL tip.
- Air dry by leting tube sit for 2 minutes with lid open until EtOH is gone.
- Resuspend in 25 µL Low EDTA TE Buffer.
- Remove from magnet, flick bead down, vortex + spin down. Make sure pellet is not sticking to side of tube.
- Let sit for 5 minutes.
- Place back on magnet until clear (~ 3 minutes).
- Transfer cleaned ligation product to new tube. TOSS BEADS
Materials Needed:
- molecular grade H20
- 5X Kappa HiFi Buffer
- 10µM dNTP
- Kappa HiFi Polymerase (HotStart)
- 5 µM *N-iTru5 Primer
- need to make 5 µM primer beforehand (stock is at at 100 µM)
- 3.75 µL Stock & 71.25 µL water
Note: At this point, I take all of the materials out and place into a cooling block in the fridge except for the HotStart for easy assess.
Master Mix
| PCR Mix | 1X | 5X |
|---|---|---|
| dH2O | 29.5 µL | 147.5 µL |
| Kappa Buffer | 10 µL | 50 µL |
| iTru5 Primer | 3 µL | 15 µL |
| dNTP | 1.5 µL | 7.5 µL |
| Kappa HiFi Polymerase | 1.0 | 5.0 |
| Total Volume | 45 µL | 225 µL |
vortex master mix upon completion
- Add 45 µL of master mix to four PCR tubes.
- Add 5 µL pooled DNA to each PCR tube.
- Spin down tubes.
Per Tube Total: 50 µL
- Incubate samples with heated lid
- 95 °C for 2:00
- 60 °C for 0:30
- 72 °C for 5:00
- Spin down PCR product after removing from thermal cycler.
- Pool PCR products in one tube.
Tube Total: 200 µL
- Repeat steps for Bead Cleanup #1 but use 1.5X beads
i.e. 300 µL of Beads
Materials Needed:
- molecular grade H20
- 5X Kappa HiFi Buffer
- 5 µM iTru7 Primer
- need to make 5 µM primer beforehand (stock is at 100 µM)
- see note in step 6a about iTru7 Primer(s)
- 5 µM P5 Primer
- need to make 5 µM primer beforehand (stock is at 100 µM)
- 10µM dNTP
- Kappa HiFi Polymerase (HotStart)
Master Mix
| PCR Mix | 1X | 5X |
|---|---|---|
| dH2O | 26.5 µL | 132.5 µL |
| Kappa Buffer | 10 µL | 50 µL |
| P5 Primer | 3 µL | 15 µL |
| dNTP | 1.5 | 7.5 |
| Kappa HiFi Polymerase | 1.0 | 5.0 |
| Total Volume | 42 µL | 210 µL |
vortex master mix upon completion
- Add 42 µL into four PCR tubes.
- Add 3 µL of one iTru7 Primer to one tube (repeat four times - see below).
- For the iTru7 Primer, you will add one indexed primer to one tube, respectively. I.e., i7 101-1 will go into tube 1, i7 101-2 will go into tube 2, etc.
- WRITE IN NOTEBOOK WHAT iTru7 PRIMERS YOU USED!!
- The above statement approach may vary if multiplexing more than two plates on a single run.
- Make sure the primer combination you decide to use is suitable for your multiplexing strategy on a NovaSeq.
- Add 5 µL pooled 8N PCR product to each tube, then spin down.
Per Tube Total: 50 µL
- Incubate samples with heated lid
- 95 °C for 3:00
- 6 cycles of:
- 98 °C for 0:20
- 60 °C for 0:15
- 72 °C for 0:30
- 72 °C for 5:00
- Spin down PCR product after removing from thermal cycler.
- Pool PCR products in one tube.
- Repeat steps for Bead Cleanup #1 but use 2X beads and resuspend in 35µL of Low EDTA TE Buffer
i.e. 400 µL of Beads
- Size select DNA using BluePippin -Follow BluePippen instructions. -Be sure to use 200µL pipette tip, not 300µL when pipetting off buffer from cassette.
Note: blue pippen cassettes can be stored for re-use of unused lanes
Materials Needed:
- molecular grade H20
- 5X Kappa HiFi Buffer
- 5 µM P5 Primer
- 5 µM P7 Primer
- need to make 5 µM primer beforehand (stock is at 100 µM)
- 10µM dNTP
- Kappa HiFi Polymerase (HotStart)
Master Mix
| PCR Mix | 1X | 5X |
|---|---|---|
| dH2O | 26.5 µL | 132.5 µL |
| Kappa Buffer | 10 µL | 50 µL |
| P5 Primer | 3 µL | 15 µL |
| P7 Primer | 3 µL | 15 µL |
| dNTP | 1.5 | 7.5 |
| Kappa HiFi Polymerase | 1.0 | 5.0 |
| Total Volume | 45 µL | 225 µL |
vortex master mix upon completion
- Add 45 µL into four PCR tubes.
- Add 5 µL pooled 8N PCR product to each tube, then spin down.
Per Tube Total: 50 µL
- Incubate samples with heated lid
- 95 °C for 3:00
- 6 cycles of:
- 98 °C for 0:20
- 60 °C for 0:15
- 72 °C for 0:45
- 72 °C for 5:00
- Spin down PCR product after removing from thermal cycler.
- Pool PCR products in one tube.
- Repeat steps for Bead Cleanup #1 but use 1X beads
i.e. 200 µL of Beads
- Re-suspend in 25 µL 10 mM TrisHCL buffer.
Note: 100 µL TrisHCL + 9,900 µL H20 = 10 mL of 10 mM TrisHCL